Density Of Silver Grains Research Articles

Developmental Changes in Hypothalamic Kiss1 Expression during Activation of the Pulsatile Release of Luteinising Hormone in Maturing Ewe Lambs

Onset of puberty is characterised by a marked increase in the frequency of release of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH). The Kiss1 gene plays a critical role in pubertal development, and its product, kisspeptin, stimulates GnRH and LH release. In the present study, we tested the hypothesis that Kiss1 gene expression in the preoptic area (POA) and hypothalamus increases during maturation of the reproductive neuroendocrine axis in association with increased LH pulsatility. Ovariectomised, oestradiol-replaced lambs were euthanised at 25, 30 and 35 weeks of age. Blood samples were collected before euthanasia to characterise the pattern of LH release. Kiss1 mRNA was detected in coronal sections of the POA and hypothalamus and Kiss1-expressing cells were identified on the basis of silver grain density. The mean number of Kiss1-expressing cells in the POA/periventricular (PeV) areas increased from 25 to 30 weeks of age. No further increase at 35 weeks of age was observed, and the changes in Kiss1 expression in the POA/PeV were independent of changes in LH pulse frequency. The mean number of Kiss1-expressing cells in the arcuate (ARC) nucleus did not differ among age groups, although it was greater in the middle ARC of lambs exhibiting increased frequency of LH release. The density of silver grains per cell did not differ among groups in any of the areas studied. The results obtained indicate that the Kiss1 gene is activated in the POA/PeV and ARC of ewe lambs during juvenile development, and that kisspeptin neurones in the middle ARC, in particular, are involved in the acceleration of pulsatile LH release during maturation of the reproductive neuroendocrine axis in ewe lambs.

Regulation of Cocaine‐ and Amphetamine‐Regulated Transcript‐Synthesising Neurons of the Hypothalamic Paraventricular Nucleus by Endotoxin; Implications for Lipopolysaccharide‐Induced Regulation of Energy Homeostasis

Infectious diseases and the administration of bacterial lipopolysaccharide (LPS) result in decreased food intake and increased energy expenditure. Because the hypothalamic paraventricular nucleus (PVN) has pivotal roles in the regulation of energy homeostasis and expresses an anorexic peptide, cocaine- and amphetamine-regulated transcript (CART), we hypothesised that increased CART synthesis in this nucleus may contribute to LPS-induced changes in energy homeostasis. Therefore, we studied the effects of intraperitoneal administration of LPS on CART gene expression in the PVN by semiquantitative in situ hybridisation. LPS caused a rapid increase in CART mRNA levels in the PVN. One hour after treatment, the density of silver grains was increased by three-fold in the PVN, and remained elevated 3 h after treatment. Because the dorsal vagal complex, an important vegetative centre in the brainstem, is heavily innervated by CART-containing axons, we determined whether the retrograde tracer, cholera toxin B subunit (CTB), accumulates in CART neurons in the PVN following stereotaxic injection of the tracer into the dorsal vagal complex. One week after injection, CTB accumulated in CART neurons in the ventral, medial, and lateral parvocellular subdivisions of the PVN. In addition, LPS administration induced c-fos expression in a population of CART neurons in the PVN that project to the dorsal vagal complex. These data indicate that increased CART gene expression in neurons of PVN may contribute to LPS-induced anorexia, and suggest that this action may be mediated, at least in part, through a PVN-dorsal vagal complex pathway.

Developmental expression of genes involved in neurosteroidogenesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in the rat brain.

In the central nervous system, neurosteroids, in particular progesterone, have neurotrophic and neuroprotective effects. We thus decided to study the developmental expression of 3beta-hydroxysteroid-dehydrogenase/Delta(5)-Delta(4) isomerase (3betaHSD), an enzyme that converts pregnenolone to progesterone, in the male rat brain at 0, 7, 14, and 70 d after birth. 3betaHSD mRNA was widely distributed throughout the brain, as shown by in situ hybridization. At all ages, the same cerebral structures were labeled, but the intensity of the hybridization signal constantly decreased during postnatal development. As the hippocampus is of particular interest because of its neuronal plasticity, we chose to quantify the changes in 3betaHSD mRNA levels as well as progesterone and pregnenolone concentrations in this structure. Quantitative in situ hybridization confirmed a decrease in the expression of 3betaHSD mRNA with progressing age, as revealed by a significant reduction in the density of silver grains per cell in the CA1 layer. This decrease was confirmed by semiquantitative RT-PCR on hippocampal samples. Concentrations of hippocampal pregnenolone and progesterone measured by gas chromatography/mass spectrometry were highest on the day of birth and lower at the other ages. Plasma concentrations of these steroids were lower than those in the hippocampus, suggesting that they may have been mostly synthesized in situ since the day of birth. These results demonstrate variations in the expression of a gene coding for an enzyme critically involved in progesterone synthesis in the hippocampus throughout postnatal development.

Evaluation of the cholesterol influence in type II collagen-induced arthritis in DBA/1J mice: an autoradiographic study.

In order to verify the cholesterol influence in RA severity in DBA/1J mice, we quantified the cholesterol present in the knee joints of normal (N) and with collagen II induced arthritis (CIA). Forty male DBA/1J mice, were divided in normal (n=20) and CIA group (n=20). Mice in CIA group were injected with 100 microg of collagen II emulsified in Freund's complete adjuvant. Sixteen DBA/1J (8 N and 8 CIA) received an injection of 2.96 x 10(6) Bq of (3)H-cholesterol and were anesthetized and sacrificed. Semi-fine sections were covered with LM-1 emulsion, exposed for six weeks and developed. Collagen induced edema, erythema and dysfunction of knee joints in CIA group. Radioactive cholesterol was located more on the synovial membrane, where we found the greatest density of silver grains, significantly (P<0.0001) higher in group CIA vs. controls (61-/+2.3 X 18-/+0.7). We conclude that the cholesterol deposits on the synovial membrane is related to CIA severity.

A method to measure the effective spread of focally injected muscimol into the central nervous system with electrophysiology and light microscopy

A method was developed to quantitate the volume of brain inactivated by muscimol focally injected. Tritiated muscimol was injected into the cerebellum and closely spaced sequential microelectrode recordings made at different depths by penetrations in an X–Y pattern centered at the injection site to evaluate changes in spontaneous activity in the tissue volume. Animals were euthanized after survivals from 40 min to 6 h, the cerebellum sectioned in the sagittal plane, and the sections dried onto glass slides. The slides were dipped in photographic emulsion, exposed in the dark and developed. Silver grain densities were quantitated by light microscopy from measured standards. The extent and concentration of bound, labeled muscimol co-varied with the observed reduction in recorded spontaneous activity. For future studies, the distribution and density of silver grains alone can serve as an accurate spatial indicator of the area of muscimol inactivation at high spatial resolution.

Galanin modulates the activity of proopiomelanocortin neurons in the isolated mediobasal hypothalamus of the male rat

It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as β-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on β-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous β-endorphin release within the first 30–60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on β-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in β-endorphin release. Measurement of β-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of β-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.

NEURONAL POPULATIONS OF RAT CEREBRAL CORTEX AND HIPPOCAMPUS EXPRESSED A HIGHER DENSITY OF L-TYPE Ca2+ CHANNEL THAN CORRESPONDING CEREBRAL VESSELS

Dihydropyridine (DHP)-type Ca2+ antagonists block primarily L-type Ca2+ channels and are used in the therapy of hypertension. They were also proposed for the treatment of several central nervous system disorders. In brain, these compounds bind both neuronal and vascular Ca2+ channels, but no studies have evaluated comparatively their density at neuronal and vascular level. This study has analyzed the pharmacological profile and the anatomical localization of L-type Ca2+ channels in rat frontal cortex, hippocampus and in forebrain pial and intracerebral arteries by radioligand binding assay and high resolution light microscope autoradiography. The DHP derivative [3H]nicardipine was used as a radioligand. Binding of [3H]nicardipine was consistent with the labeling of L-type Ca2+ channels. In frontal cortex, the highest density of binding sites was found in nerve cell body region, followed by the neuropil and the wall of intracerebral arteries.In hippocampus, the density of binding sites was higher in the nerve cell body region than in the neuropil of CA1, CA3, and CA4 subfields. In the dentate gyrus, a higher density of silver grains was developed in neuropil than in nerve cell body of granule neurons. With the exception of dentate gyrus, neuronal binding sites were more expressed than vascular binding sites in the hippocampus. In pial arteries [3H]nicardipine binding density decreased concomitant with the reduction of vessel diameter, whereas in intracerebral arteries [3H]nicardipine binding density displayed an opposite pattern. The above findings indicate that in brain the density of neuronal L-type Ca2+channels was significantly higher than that of vascular ones. This may account for more pronounced neuronal than vascular effects after pharmacological manipulation of cerebral Ca2+ channels.

Processing the spinal cord for in situ hybridization with radiolabelled oligonucleotides

Publisher Summary The spinal cord receives sensory information from the trunk and limbs and is the final station for movement commands. Its anatomy and physiology are well defined and detailed knowledge about the segmental spinal-cord circuitry makes this structure ideal for experimental studies. As the first station to integrate somatosensory information in the central nervous system (CNS), the spinal cord is of outstanding interest to the field of pain research and therapy, and is extensively studied in models of traumatic injury or regeneration. This chapter describes the application of the in situ hybridization (ISH) method to investigate gene expression in the spinal cord of the adult rodent. This method has been used extensively to investigate the expression of neurotransmitters and to characterize neuroplastic changes following noxious sensory inputs in various experimental pain-model systems in the spinal cord. The ISH signal in the spinal cord can be quantified macroscopically from X-ray autoradiographs and in cellular resolution from emulsion-dipped sections. Quantification of silver-grain density in emulsion-dipped sections is a much more accurate, semiquantitative measure of the mRNA amount labeled because this quantification is selective for the ISH label over defined cells or structures.

Ornithine metabolism along the female mouse nephron: localization of ornithine decarboxylase and ornithine aminotransferase.

The fate of ornithine in the nephron of the female OF-1 Swiss mouse remains unknown. The aim of the present study was to identify the nephron segments containing the key enzymes involved in ornithine metabolism: ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). Viable tubules isolated by microdissection were incubated with [1-14C]ornithine to study the oxidative pathway. Other tubules were permeabilized to measure the ODC activity. Ornithine was decarboxylated in all intact tubules. Gabaculine, a suicide inhibitor of OAT, and rotenone sharply decreased the production of 14CO2 from [1-14C]ornithine. No ODC activity was found in permeabilized tubules isolated from untreated mice. Testosterone increased ODC activity in the proximal tubule substantially and to a minor extent in other nephron segments. In situ hybridization showed ODC messenger ribonucleic acid (mRNA) to be absent in kidneys of untreated females but abundant in the cortex and the outer stripe of the outer medulla of testosterone-treated female mice. The whole proximal tubule contained a great density of silver grains corresponding to ODC mRNA. In conclusion, no basal ODC activity was found in the nephron of female mice. The testosterone-inducible ODC is localized mainly in the proximal tubule, but is also present in distal tubules and collecting ducts. OAT is distributed along the whole nephron, but its activity is higher in proximal tubules than in distal tubules.

Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation.

We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.

Localization of a sodium-dependent high-affinity serotonin transporter and recruitment of exogenous serotonin by the cestodeHymenolepis diminuta: an autoradiographic and immunohistochemical study

Uptake of serotonin by tissues of intact and hemitransected Hymenolepis diminuta was studied by autoradiography and immunohistochemistry. Hemitransected worms were incubated in balanced saline containing 10 µM [3H]serotonin and washed extensively. The density of silver grains over the serotonin-immunoreactive longitudinal nerve cords and commissural rings, male reproductive system, tissues surrounding the genital pouch, and deep longitudinal muscles was significantly greater than that over the parenchyma. The presence of serotonin in spermatozoa suggested a role for this amine in spermatozoon activity. In contrast, uptake of 10 µM [3H]serotonin in sodium-free saline was significantly reduced compared with that in balanced saline in all tissues examined except the parenchyma. Analysis of the data revealed that the sodium-dependent high-affinity serotonin transport system is localized primarily in the serotonergic-like neurons of H. diminuta, which suggests possible recycling of neuronally released serotonin. Following incubation of intact worms in vitro for 12 h in 5 µM [3H]serotonin, the density of silver grains was significantly higher over the serotonin-immunoreactive nerves, elements of the male reproductive system, tissues surrounding the genital pouch, and deep longitudinal muscles than over the parenchyma. These results demonstrate recruitment of exogenous serotonin by intact H. diminuta and suggest sequestration and concentration by the serotonin-immunoreactive neurons via the sodium-dependent high-affinity transporter. These data further suggest that although H. diminuta can synthesize serotonin, it may obtain serotonin from the host. Nonetheless, the amount of serotonin recruited by H. diminuta from the host in vivo compared with that which they synthesize is not known.

Autoradiographic localisation of muscarinic cholinergic receptor subtypes in human placenta

The localisation of M1–M4 muscarinic cholinergic receptor subtypes was investigated in sections of normal human term placenta by light microscope autoradiography. Muscarinic cholinergic receptor subtypes were found almost exclusively in syncytiotrophoblast. Neither other cellular components of placenta, nor blood vessels were labelled. Quantitative analysis of the density of silver grains developed in sections incubated with the different protocols for labelling M 1–M 4 receptor subtypes, revealed that syncytiotrophoblast expresses all subtypes of muscarinic cholinergic receptor investigated. A higher density of binding sites was found in the apical than in the basal portion of syncytiotrophoblast. The demonstration of muscarinic cholinergic receptors in syncytiotrophoblast suggests that a cholinergic system may have a role in regulating transport of compounds from maternal to foetal interface.

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart.

1. The pharmacological profile and the anatomical localization of a putative dopamine D4 receptor were assessed in sections of rat and human atria and ventricles using combined radioligand binding and autoradiographic techniques with [3H]-spiperone as a ligand. 2. [3H]-Spiperone was bound specifically to sections of human and rat atria and ventricles. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high-affinity sites. In atria, the dissociation constant value (Kd) was 0.45 nM in rats and 0.32 nM in humans, and the maximum density of binding sites (Bmax) was 31.6+/-2.9 fmol mg(-1) tissue in rats and 18.8+/-0.7 fmol mg(-1) tissue in humans. In ventricles, Kd was 0.38 nM in rats and 0.39 nM in humans, and the Bmax was 43.5+/-3.0 fmol mg(-1) tissue in rats and 56.4+/-3.2 fmol mg(-1) tissue in humans. 3. The pharmacological profile of [3H]-spiperone binding to sections of both rat and human atria and ventricles was consistent with the labelling of dopamine D2-like receptors. [3H]-Spiperone binding was more sensitive to displacement by the neuroleptic clozapine in sections of atria than of ventricles, suggesting the expression of a dopamine D4 receptor in atrial tissue. Moreover, preincubation of some sections with a dopamine D4 receptor antibody and subsequent exposure to [3H]-spiperone caused a remarkable decrease of radioligand binding to sections of atria, but only a slight reduction of binding to sections of ventricles. 4. Light microscope autoradiography revealed the accumulation of silver grains over atrial tissue within atrial myocardiocytes. A higher density of silver grains was developed in rat than in human atria. In ventricles, silver grains were accumulated primarily in cluster areas both in rats and in humans. 5. The above findings suggest the expression of a dopamine D4 receptor in rat atria, but not in ventricles. A similar site was identified in human atria. The possible relevance of a dopamine D4 receptor in the heart is discussed.

Proopiomelanocortin (POMC) mRNA expression: distribution and region-specific down-regulation by chronic morphine in female guinea pig hypothalamus

There is compelling evidence that endogenous opioid peptides are regulated by exogenous opiates. Our previous studies have shown that the μ-opioid receptor protein and mRNA are down-regulated in the mediobasal hypothalamus of the female guinea pig following chronic morphine treatment. In addition, electrophysiological studies have shown that hypothalamic β-endorphin ( β-EP) neurons express μ-opioid receptors that are uncoupled and down-regulated following chronic morphine treatment. Currently, we tested the hypothesis that chronic morphine, which produces down-regulation of μ-opioid receptors, causes a down-regulation of pro-opiomelanocortin (POMC, the precursor of β-EP) mRNA expression in female guinea pig hypothalamus. Female guinea pigs were ovariectomized and implanted subcutaneously (s.c.) with 4×75 mg pellets for 2 days plus six more pellets of either morphine ( n=6) or placebo ( n=6) for another 5 days. Animals were sacrificed between 1000 and 1100 h on day 7. The expression of POMC mRNA were investigated using in situ hybridization histochemistry with a guinea pig specific 35S-labeled cRNA probe in hypothalamic tissue sections. POMC mRNA was localized to the arcuate nucleus (Arc) and median eminence (ME) of the medial basal hypothalamus. The distribution pattern was the same in both morphine and placebo control animals. However, the density of silver grains was less in morphine treated animals versus placebo control animals. Overall, the level of POMC mRNA was decreased by 22% in the Arc of morphine-treated guinea pigs as compared with the placebo controls ( p<0.05). This decrease in POMC mRNA expression was even greater in the caudal Arc (28%, p<0.01) in morphine-treated animals. These results suggested that the biosynthetic activity of POMC neurons is down-regulated with chronic exposure to morphine.

変形時にアルミニウムおよびチタン合金から放出される微量不純物水素の水素マイクロプリント法による可視化

Hydrogen microprint technique with the aid of photographic effect was applied to pure aluminum and three types of titanium alloys to visualize the hydrogen atoms evolved during tensile deformation. Tensile specimens covered with a monolayer of photographic emulsion were deformed by about 5% at temperatures ranging from −180 to 82°C, subsequently developed and fixed under the safety light in a dark room. Silver grains indicating hydrogen emanation were revealed along coarse slip lines and some grain boundaries, and at second phase (constituent) particles. The density of silver grains was affected by the impurity hydrogen content of the specimen and by deformation temperature. Consequently it can be concluded that hydrogen atoms in the interior of the specimen are transported to the surface with a number of gliding dislocations at room temperature, and that grain boundary becomes a primary diffusion path at elevated temperatures. The newly developed technique with the aid of photographic effect has a higher sensitivity to hydrogen emanation than the conventional hydrogen microprint technique. Moreover, the present technique is by far simpler than tritium autoradiograhy and can be applied to a wide variety of metals and alloys which deform primarily by dislocation glide.

Tissue uptake and interconversion of plasma unesterified 14C linoleic acid in the guinea pig

Part of the arachidonic acid (20 : 4, n−6) pools in the gastrointestinal tract and blood forming tissues may be formed by local interconversion of linoleic acid (18 : 2, n−6) taken up as a free fatty acid from blood. This study examined the rate of uptake and interconversion of unesterified 14C-18 : 2 by different tissues in young guinea pigs. The clearance rate of 14C-18 : 2 was fast, and the initial half-life was 6.3 s. The retention of 14C in tissue lipids was 1.6–1.8% g −1 in the liver, 0.4% g −1 in stomach, 0.7% g −1 in small intestine, 0.2% g −1 in colon, 0.4% g −1 in bone marrow and 0.7% g −1 in spleen. Autoradiographic localization of 3H-18 : 2 under light microscope demonstrated that most of the 3H radioactivity of the gastrointestinal tract was in the mucosa, in both villus and crypt cells. In bone marrow smears, a high density of silver grains was found in megakaryocytes. The percent of 14C in δ6 desaturase products was higher in gastrointestinal tract, heart, lung, bone marrow and spleen than in liver. The ratio of 14C-20 : 3 / 14C-20 : 4 formation in most tissues was high, and a notable finding being a lower rate of 20 : 4 formation from plasma free 18 : 2 in the liver, (170 pmol min −1) than in the gastrointestinal tract (428 pmol min −1) and bone marrow (1203 pmol min −1). The local interconversion of 18 : 2 into δ6 desaturase products is thus an important source of 20 : 4 in these organs in guinea pigs.

The neuropeptide Y (Y4) receptor is highly expressed in neurones of the rat dorsal vagal complex.

Recently, the cDNA encoding the Y4 neuropeptide Y (NPY) receptor cDNA was cloned from a rat genomic library. The Y4 receptor is characterized by having a high affinity for pancreatic polypeptide (PP) and peptide YY (PYY). By using in situ hybridization histochemistry with 35S-labelled riboprobes, we have visualized the cellular expression of mRNA encoding the Y4 receptor protein in the rat dorsal vagal complex at the light microscopical level. High densities of silver grains were observed over neurones of the dorsal vagal motor nucleus, and over neurones of a subregion of the nucleus of the solitary tract known as the subnucleus gelatinosus. Furthermore, cells within the ventral margin of the area postrema expressed high levels of Y4 mRNA. These observations indicate that circulating PP and/or NPY/PYY via the blood-brain barrier-free area postrema and subpostremal area could influence neurones of the dorsal vagal complex with profound influence on numerous homeostatic mechanisms governed by this nuclear complex.

Towards quantitative in situ hybridization.

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue.

The effect of hypophysectomy and growth hormone replacement on sst1 and sst2 somatostatin receptor subtype messenger ribonucleic acids in the arcuate nucleus.

Although considerable evidence indicates that somatostatin (SRIF) exerts direct actions on GH-releasing hormone-containing arcuate neurons within the hypothalamus to modulate hypophyseal GH secretion, the underlying mechanism(s) remains to be elucidated. We recently demonstrated high levels of expression of the messenger RNAs (mRNAs) coding for two prototypic receptors of the recently cloned SRIF receptor (sst) family, sst1 and sst2, in the arcuate nucleus of the rat hypothalamus. However, information on the biological roles of these receptor subtypes and the factors regulating their expression is lacking. In the present study, we hypothesized that perturbations in GH would influence sst mRNA levels in cells of the arcuate nucleus in vivo. To test this hypothesis, we examined the effects of hypophysectomy (HPX) and HPX with GH replacement, on sst1 and sst2 mRNA levels in the brains of adult male rats by in situ hybridization using 35S-labeled antisense riboprobes. The number of labeled cells and the density of silver grains per cell were quantified using a computer-assisted image analysis system. Two weeks after HPX, there was a 50-60% reduction in both the number and labeling density of sst1 and sst2 mRNA-expressing cells in the arcuate nucleus compared to those in sham-operated control rats. Administration of recombinant human GH (200 micrograms/day for 7 days by continuous sc infusion using osmotic minipumps) to HPX rats augmented both the cell number (P < 0.05) and labeling density (P < 0.01) of sst1 mRNA in the arcuate nucleus, but did not significantly alter sst2 mRNA levels compared to those in HPX rats infused with H2O. There were no significant changes in sst1 and sst2 mRNA levels in extra-arcuate areas, including the cerebral cortex and medial habenula, or in suprachiasmatic, medial preoptic, and magnocellular preoptic nuclei after either HPX or GH replacement. These results indicate that the expression of both sst1 and sst2 SRIF receptor subtypes in brain is under the regulatory influence of pituitary hormones. They further suggest that GH may participate in the regulation of its own secretion by influencing the expression of the sst1 receptor gene on arcuate neurons. Such a mechanism may be important in the feedback regulation of GH secretion by the arcuate nucleus.

Accumulation of Hg(II) Ions in Mouse Adrenal Gland.

Female BALBc mice were administered HgCl2 at a single dose of 4 mg/kg i.p. The acute intoxication with Hg(II) salts (2 hr) caused accumulation of Hg(II) ions in the adrenal gland in general, and in the medulla, in particular. Based on data obtained with atomic absorption spectroscopy and quantitative cytochemistry, we determined the amount of mercury (II) in the adrenal glands and found it to be 14.2 ng Hg(II) (3.5 mg/kg wet weight of the adrenals). An uneven distribution of Hg(II) was found within the adrenal gland, not only between the medulla and cortex, but also within the cortex. The applied autometallographic method revealed that the cortex was negative except the zona glomerulosa, whereas the medulla showed a strong reaction localised to the chromaffin granules of the secretory cells. Both adrenaline and noradrenaline producing cells reacted. The comparison of the density of silver grains by scanning densitometry in the medulla and cortex revealed a significantly higher Hg(II) concentration in the medulla compared to the cortex (10 mg/kg vs 2 mg/kg, respectively). The results presented here suggest that there may be a connection between the symptoms of acute Hg(II) intoxication and its adrenal accumulation.